Fibroproliferative diseases of the lung and other organs constitute a heavy burden of morbidity and untimely deaths. Fibrosis results in permanent loss of the tissue's ability to function optimally. In the lung, gas exchange ability is impaired by the formation of scar tissue. Often, Idiopathic Pulmonary Fibrosis (IPF) is diagnosed late (on CT scans/lung biopsy) and has a high mortality with a median survival time from diagnosis of 3 years. It is currently impossible to identify patients with Adult Respiratory Distress Syndrome (ARDS) and other inflammatory lung diseases that are developing secondary fibrosis. Moreover there are no effective therapies for fibrosis despite it being a highly active cellular process which should be accessible to intervention. Part of the problem is the time required to establish drug effectiveness in vivo and the poor utility of existing biomarkers. Thus, there is an urgent need to develop diagnostic methodologies that will permit the more effective and rapid determination of both disease activity and efficacy of emerging anti-fibrotic drugs.
Molecular imaging offers a viable approach to interrogate non-invasively living samples in real time with spatial resolution when combining the technical instrumentation with optical imaging probes. Optical probes are typically developed for each specific target for effective diagnostic imaging (Biochemistry 2010, 49, 1364-1376). Activatable optical probes can provide functional details of molecular events, and provide advantages such as providing information at the molecular level. Furthermore, they can be used with a small portable system, provide information quickly, and can be microdosed (<100 μg), thereby reducing the risk of side effects.
fCFM (fibre-Confocal Fluorescence Microscopy) has been used for studying in vivo the alveolar structure of the human lung during bronchoscopy [Eur. Resp. J 2009, 33, 974-985. Proc. Am. Thorac. Soc. 2009, 4, 444-449]. Its use in combination with optical probes for specific enzymes can provide valuable information.
Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases capable of degrading the extracellular matrix. MMPs have long been of interest as pharmaceutical targets and play an important role in the tissue remodelling associated with various physiological or pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis, and metastasis (Cancer Metastasis Rev. 2008, 27, 679-690). They can also be considered as biomarkers overexpressed in fibrotic lung [Eur. Respir. J. 2011, 38 (6) 1461-1467; Am. J. Respir. Crit. Care Med 2000, 162(5) 1949-1956, Eur. Respir. J. 2009, 33, 77-84] making them ideal for molecular targeting with labelled peptides and could serve as a useful tool for early diagnosis of diseases via optical imaging. Over-expression and knockout studies in mice show a critical role of proteinases (MMP-2/9) in lung fibrosis, and their expression has been consistently shown to be elevated in the lung lavage fluid of fibrotic subjects compared both to levels in healthy lung and levels in patients with other lung diseases [Cell Biol. Toxicol. 2002, 18(1), 51-61].
Many proteinase probes exploit the FRET (Forster Resonance Energy Transfer, also known as Fluorescence Resonance Energy Transfer) phenomenon to detect enzymatic activity [Biotechnol. J. 2014, 9, 266-281, Chem. Comm., 2008, 4250-4260], where the protease substrate is located between a fluorophore/quencher pair. Alternatively, the “self-quenching” effect in multi-branched systems previously described for the detection of AspN Endoproteinase [Angew. Chem. 2002, 41, 17, 3233-3236] and more recently HNE [Org. Biomol. Chem., 2013, 11, 4414-4418] has been applied as well for cathepsin S [J. Med Chem. 2006, 49, 4715-4720].
Several matrix metalloproteinase probes have been described over the years based on potent MMP inhibitors and activatable fluorescent probes. To date, design and development of labelled substrates for these enzymes have largely focused on sensing tumour-related activity [review Cancer biotherapy and Radiopharmaceuticals, 21, 5, 2006, 409-416], as well as for osteoarthritis or atherosclerosis [Chem. BioChem. 2012, 13, 2002-2020; Contrast Media Mol. Imaging 2014, 9, 187-210]. Despite many efforts, most of the probes known in the art are limited in usefulness in vivo at least, due to poor specificity and in vivo stability.
Accordingly, it is an object of the present invention to provide improved optical probes that are capable of specifically detecting MMP, or types of MMP, that are preferably stable in vivo.